A number of recent pan-cancer studies have demonstrated the importance of the tumor's tissue of origin in determining its mutational signature. Carcinogenesis is also characterized by altered metabolism wherein cancer cells rely primarily on aerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production, even in the presence of oxygen. However, the precise metabolic profiles of tumor cells are not well studied with respect to their tissue of origin. In the context of normal cells, tissue types such as muscle, liver, and kidney utilize fatty acid oxidation for mitochondrial energy production, harbor higher mitochondrial-encoded gene expression levels, and produce substantially higher levels of ATP relative to their counterparts that utilize glucose as their primary substrate. Although there is an accumulation of somatic mitochondrial DNA (mtDNA) mutations in these tissue types, the impact of these mutations on the role of mitochondria in reprogramming cancer cell bioenergetics is unclear. I hypothesize that tissue types with high demands for mitochondrial ATP production are more prone to increased somatic mtDNA mutations and differential mitochondrial gene expression, and that this contributes to altered energy metabolism and increased tumorigenic potential in their cancer cells. To address this notion, I propose to mine roughly 8,000 next-generation sequencing (NGS) datasets across 14 cancer types from the Cancer Genome Atlas (TCGA) to uncover tissue- and tumor-specific mitochondrial sequence variants. My preliminary analysis of TCGA whole-genome sequencing data from 905 patients lends strong support for a tissue-specific signature of the mitochondrial genome in neoplastic cells, with prominent aberrations in mitochondria of the muscle, liver, and kidney tissues. Hepatocellular carcinomas carry an elevated mtDNA mutation burden and are enriched for non-synonymous variants. Additionally, patients with sarcoma, and hepatocellular and renal carcinomas present with severe depletion of their tumor mtDNA content, unlike the remaining cancer types. Furthermore, preliminary analysis of RNA-sequencing data from roughly 1,825 patients suggests that mitochondrial-encoded gene expression clusters distinctly based on the tumor's tissue of origin. To test the impact of altered mitochondrial genetic content on cancer progression, I propose to construct cytoplasmic hybrids (cybrids) containing mitochondrial sequence variants in cancer cell lines and assess their effect on nuclear gene expression, cellular bioenergetics, and cancer hallmarks in vitro. Using cybrids controls for nuclear genetic content and allows the identification of phenotypic changes that can be directly attributed to the cell's mitochondrial genetic content. To investigate the role of mitochondria in cancer, my proposed research objectives are two-fold: (1) identify changes in the mitochondrial genome and transcriptome within and across cancer types and (2) assess their impact on cancer progression in vitro using cybrid technology.